Characterization of in vitro transcription initiation and termination sites in Col E1 DNA

Overlapping restriction fragments from the region between the single Eco R1 site and the origin of replication of the plasmid, Col E1, have been utilised as templates in an in vitro transcription assay using E. coli RNA polymerase. Transcription towards the single Eco R1 site is initiated at a point 415 bp to the origin side of that site. In vivo, transcription starting at this point probably produces the mRNA for the colicin immunity protein. Transcription away from the Eco R1 site is initiated at a point 140 bp to the origin side of that site and terminated 30 bp further on. This terminator is probably the point at which transcription of the colicin gene is terminated in vivo. DNA sequence analysis in both these regions demonstrated several similarities to other prokaryotic regulatory regions. 50% homology between the putative immunity promoter and other prokaryotic promoters is apparent, so are similarities in AT-content. Upstream of the ATG start codon the sequence PuPuTTTPuPu and a termination codon (TAA) appear; both are typical of prokaryotic ribosome binding sites. The colicin terminator demonstrated similarities to other rho-independent prokaryotic terminators: a GC-rich region with termination in an adjacent AT-rich region containing T clusters on the non-coding strand. The possible role of initiation upstream from the colicin terminator is discussed.